Cholera vaccines

ABSTRACT

The gene encoding the TcpA pilus has been cloned. It encodes a protein useful in live, killed-cell, and synthetic vaccines. Protein production is enhanced by specific medium conditions.

BACKGROUND OF THE INVENTION

This application is a continuation of Mekalanos, et al., entitled"Cholera Vaccines" U.S. Ser. No 07/188,016, filed Apr. 29, 1987 U.S.Pat. No. 5,098,998, now abandoned which is a continuation-in-part ofU.S. Ser. No. 07/043,907 filed Apr. 29, 1987 assigned to the sameassignee as the present application and the whole specification andfigure of which is hereby incorporated by reference.

This invention relates to immunological protection against Vibriocholerae.

V. choleras is a bacterial species that can cause a diarrheal disease inhumans by colonizing in the small intestine and secreting a proteintoxin. The action of the cholera toxin has been well characterized. Thetoxin includes two subunits, the A and B subunits; the B subunit has noknown toxic activity but provides a degree of immunological protectionwhen used as a component of a vaccine. Black et al. 1986 In Advances inResearch on Cholera and Related Diarrheas 3:271. ed. Kuwahara et al.

In addition to B sub-unit vaccines, vaccines for cholera also includedenatured whole cholera toxin, killed whole cells of V. cholerae grownusually on a solid medium at pH 7.5-8.0, and mixtures of killed cellsand inactivated toxin molecules. Other vaccines include live attenuatedV. choleras strains which do not produce the A subunit of cholera toxin,and attenuated strains of heterologous non-Vibrio cholerae carriers,that is, non-V. cholerae strains, e.g., Salmonella typhi Ty21A havingcloned genes encoding the cholera protective 01 antigen (J. InfectiousDesease 131:553-558, 1975).

Ehara et al. (Trop. Med. 28:21, 1986; and Vaccine, 1988) describepurification of fimbriae of V. cholerae having a structural subunitprotein of about 16 kD. This protein is not stained by Coomassie blueand its haemagglutination (HA) titre is mannose sensitive. AL-Kaissi etal. (J. App. Bact. 58:221, 1985) also describe fimbriae which have amannose sensitive HA titre.

Kanoh (Nippon Saikingaku Zasshi, 36:465, 1981), Melnikova et al. (Mol.Biol. Genet. Russian 2:18, 1982), and Kanoh (Jpn. J. Bacteriol. 36:465,1981) describe sex pill of V. cholerae, encoded by plasmid genes.Holmgren et al. (Infect. Immun. 33:136, 1981) describe various E. colifimbriae.

SUMMARY OF THE INVENTION

This invention provides a V. cholerae surface protein structure called apilus, specifically a toxin-coregulated pilus ("TcpA pilus") that isinstrumental for adherence of V. cholerae to the intestine and forcolonization in the intestine. Production of this pilus is significantlyenhanced under certain laboratory culture conditions, and thus theinvention also provides a method for cultivation of V. cholerae toproduce the TcpA pilus. Further, the invention features a method forproduction of the B subunit of cholera toxin, which is co-regulated withproduction of TcpA pilus and is also useful as a vaccine component. Inaddition, we have cloned the structural gene for the TcpA pilus subunitand determined the inferred amino acid sequence of the TcpA protein. Theuse of these materials and information enables production of severaldifferent types of cholera vaccines that are able to stimulateproduction of protective antibodies that recognize the TcpA pilus andthus inhibit adherence to, and colonization of, the human intestine byV. cholerae.

In a first aspect, the invention features: 1) a purified polypeptidecomprising at least one immunogenic determinant of (or a polypeptidecross-reactive with) the TcpA pilus of V. cholerae, preferably the wholeof the TcpA pilus; 2) an oligonucleotide encoding at least fivecontiguous amino acids of the TcpA pilus of V. cholerae and thepolypeptide encoded by that oligonucleotide; and 3) an oligonucleotide,comprising 15 base pairs, having substantially the nucleic acid sequenceof a part of a gene encoding the TcpA pilus of V. cholerae.

By purified is meant that the TcpA pilus, or polypeptide forming a partof the pilus, is separated from one or more components with which itnaturally occurs, for example OmpU outer membrane protein. Preferably,the polypeptide is provided essentially free of these naturally occuringcomponents, represents greater than 50% of total protein in apreparation, and has less than 50% of the normal OmpU protein level.

By polypeptide is meant a sequence of amino acids encoding a part ofTcpA which is antigenetically active, and thus induces antibodyformation, the antibodies of which recognise naturally occuring TcpA.

In a second aspect, the invention features a method for producing avaccine, comprising growing a culture of V. cholerae cells in growthmedium under conditions in which TcpA is produced at higher levels thannormal, preferably in a medium having a pH of about 6.5, or less, orusing cells having a mutated htx gene; preferably, the vaccine is awhole cell vaccine and the method further comprises killing the cells;or the vaccine comprises a TcpA pilus, and the method further comprisesharvesting the V. cholerae and purifying the TcpA pilus from the cells;or the vaccine comprises B subunit of cholera toxin and the methodfurther comprises harvesting the V. cholerae cells and purifying the Bsubunit of cholera toxin from the cells.

In other aspects, the invention features a vaccine comprising TcpApilus, or an immunogenic determinant thereof, preferably purified asdescribed above; a heterologous (non-V. cholerae) bacterial straincomprising nucleic acid encoding an immunogenic fragment of TcpA pilus;a hybrid polypeptide comprising an loaf immunogenic determinant of TcpApilus of V. cholerae and an immunogenic determinant of the B subunit ofprotein, and the nucleic acid encoding therefor. The invention alsofeatures a bacterial cell having at least 1% total protein as TcpA.Preferably the cell lacks toxic levels of cholera toxin; and mostpreferably the cell is a Vibrio cholerae having a mutated htx gene.

DESCRIPTION OF THE PREFERRED EMBODIMENT Structure TcpA pilus

The FIGURE shows the DNA sequence, and corresponding amino acidsequence, of the gene encoding TcpA pilus in V. cholerae.

The TcpA pilus is an example of a filamentous surface component (pilus)of V. cholerae cells. When isolated from whole cells of V. cholerae 0395it has an apparent molecular weight in SDS polyacryamide gelelectrophoresis of 20.5 KD. The TcpA pilus is associated with themacromolecular filamentous structures on the surface of V. cholerae andcan be partially purified by differential centrifugation. The pilus isproduced in significant quantities only under certain environmentalconditions of growth of V. cholerae cells. For example, its productionis best at a low pH, preferably pH 6.5 or less, and is virtuallyundetectable at a pH above7.5. Further, its production is best at a lowionic strength equivalent tobetween 50-100 mM NaCl. Other factorscontrolling TcpA pilus production include the presence of amino acids inthe growth medium (e.g. LB, see below), aeration (e.g., 0.5-2 liter/minper 6 liter of medium), the type of medium (preferably liquid), andincubation temperature (preferably 25°-30° C.). In addition, the TcpApilus is only produced ata particular stage of growth of the V. choleraecells (e.g., stationary phase). These growth conditions generally arenot optimal for cellular growth; rather they are optimal for productionof TcpA pilus.

TcpA pill form bundles of filaments when purified, the protein isstained by Coomassie blue, and its haemagglutination activity is notaffected by mannose.

To measure the percentage of TcpA in a preparation, a portion of thepreparation is run in a gel, reacted with antibody to TcpA to locate theprotein band corresponding to TcpA, the band is cut from a gel, and theamount of TcpA is determined. Total protein is determined by standardprocedure, and used to calculate % TcpA

The gene encoding TcpA pilus in V. cholerae has been cloned, asdescribed below; its DNA sequence is shown in FIG. 1. The TcpA gene ischromosomallylocated. Uusing this cloned DNA, related DNA from otherstrains of V. cholerae, or synthetic DNA encoding substantially the sameamino acid sequence shown in the FIGURE, the TcpA pilus can be producedin other bacterial strains, using standard procedures. Thus, the termTcpA pilus includes proteins having amino acid modifications which donot substantially affect the antigenicity of the molecule. Similarly thegene encoding TcpA includes genes encoding for such amino acidmodifications, or for antigenic portions of TcpA. For example, the DNAcan be introduced into any standard vector and transformed intobacterial strains capable ofexpressing the TcpA pilus; an example ofthis is given below.

EXAMPLE 1: CLONING THE TcpA GENE

TnphoA is a derivative of the transposon Tn5 that retains its broad hostrange and random insertion properties but in addition can create fusionsbetween target genes and phoA, the alkaline phosphatase gene ofEscherichia coli (Manoil et al. 82 Proc. Nat'l Acad. Sci U.S.A. 8129,1986). Such gene fusions encode hybrid proteins composed of a carboxyterminal portion of alkaline phosphatase (PhoA) fused in frame to anaminoterminal portion of a target gene product. These hybrid proteinsexhibit little or no PhoA activity unless the target gene encodes asecreted or membrane spanning protein. These active fusions are easilyidentified as blue colonies by incorporating the chromogenic alkalinephosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate (XP) intoagar medium on which the bacterial cells carrying inserts of TnphoA havebeen plated. Because virtually all bacterial proteins implicated asvirulence factors are periplasmic, extracellular, or cell surfaceassociated, this method provides a strong enrichment for insertionmutations that may affect pathogenic properties of bacteria, such ascolonization or toxin production.

Random insertions of TnphoA into the chromosome of V. cholerae wasaccomplished through the use of pRT291 a derivative of the broad hostrange P-group plasmid pRK290 (Mekalanos, Nature 276:633, 1978) thatcarries a copy of TnphoA (constructed by spontaneous transposition withTnphoA). Briefly, chromosomal inserts of TnphoA were obtained in V.cholerae strains carrying pRT291 by superinfection with the incompatibleplasmid pPh1JI (I.d.) and selection for resistance to kanamycin andgentamycin. V. cholerae colonies carrying inserts of TnhphoA werescreenedfor the PhoA⁺ phenotype on LB agar containing 0.2% glucose and20 mg/ml of XP at 30° C.

A pool of several thousand V. cholerae colonies carrying inserts ofTnphoA was screened for mutations that encoded PhoA⁺ fusion proteins.About1% of the colonies carrying inserts of TnphoA were blue on thismedium indicating that they carry copies of TnphoA fused to genesencoding secreted or membrane proteins that express in this medium.

Forty of these blue colonies were purified and analyzed for their totalprotein profile by polyacrylamide gel electrophoresis (PAGE). V.cholerae wild-type and mutant strains were grown in LB broth, pH 6.5 at30° C. for 18 h with aeration. Cells were collected by centrifugation,lysed in sample buffer with a reducing agent, and analyzed byelectrophoresis through a 12.5% polyacryamide slab gel in the presenceof NaDodSO₄, as previously described by Mekalanos et al. (16 Infect.Immun. 787, 1977).

Seven mutants have detectable differences in their protein profileshowing the loss of one or more bands. One type of mutant, representedby strain RT110.21, has lost a single protein of molecular weight 20.5KD.

A mutant lacking the 20.5 KD protein was tested for colonization defectsbya competition assay involving coinfection of the mutant with theparental strain into 3-9 day old CD-1 mice essentially as described byFreter et al. 34 Infect. Inmun. 234, 1981. The mutant strain RT110.21,which had lost the 20.5 KD protein, showed a marked decrease in itsability to compete with the parental strain in vivo, but not in vitro.Other competition experiments performed with another independentlyisolated mutant lacking the 20.5 KD protein confirmed that this class ofmutant is severely defective in colonization in both infant mice as wellas adult rabbits.

Cell fractionation showed that the 20.5 KD protein is associated withmacromolecular structures that are located on the bacterial cellsurface. A nonflagellated mutant (mot-39; Mekalanos, 306 Nature 551,1983) derived from strain 0395-N1 was used to partially purify thesestructures from sheared cells by several rounds of differentialsedimentation. The strain was grown in LB broth, pH 6.5. A volume of 400ml of broth per 2 liter flask was used and the culture was incubated at150 revolutions per min for 16 h at 30° C. The cells were collected bycentrifugation and resuspended in a buffer containing 12.5 mM tris-HClpH 7.0, 25 mMNaCl, 4 μM MgCl₂, and 4 mM CaCl₂. After shearing thebacterial cells by passage through a 21 guage needle, the pill werepurified by several rounds of differential centrifugation to removecells and soluble proteins. Typically, sheared cells are centrifuged at3000×G for 20 minutes. Supernatant fluid is collected and the pelletdiscarded. The supernatant fluid is than centrifuged at 15,000×G for 40minutes. The supernatant from this step is discarded and the pelletcollected and resuspended in (TCP buffer). These two steps ofdifferential centrifugation are repeated twice again. The final pelletrepresents purified TcpA (i.e., TcpA protein represents greater than 50%of the totalcell protein) in such as preparation. The isolation of thepili was monitored by following the purification of the 20.5 kilodaltonpilus subunit by PAGE. Purified pill were observed under the electronmicroscopeafter staining preparations with 2% ammonium molybdate.Examination of these preparations in the electron microscope showed longlaterally associated fimbria or pill (7 nm in diameter). Individualpilus filaments could be seen on the surface of cells of strain 0395 butwere not seen on cells of TnphoA induced mutants that had lost the 20.5KD protein.

The 20.5 KD protein was further purified by electroelution after PAGEand subjected to N-terminal amino acid sequence analysis. The sequencedata corresponds to that shown in the FIGURE. This sequence is highlyhydrophobic and may represent part or all of a secretory signalsequence. Consistent with this conclusion, subcloning of the TnphoAfusion from strain RT110.21 and DNA sequencing has shown that the phoAgene is fused to the pilus coding sequence 92 codons downstream from thesequence encoding this hydrophobic stretch of amino acids. Two othermutants which have lost the 20.5 KD protein also have TnphoA insertsthat have fused phoA to this same open reading frame, confirming thatthis sequence does indeed represent the structural gene for the V.cholerae pilus.

We have cloned the gene for the tcpA gene in two steps. First, thetcpA-TnphoA gene fusion carried by RT110.21 was cloned onto a plasmid byselecting for its kanamycin resistant phenotype in E. coli. A gene probederived from the DNA sequences adjacent to this TnphoA fusion was thenused to identify a cosmid clone carrying the wild type tcpA gene ofstrain0395 (Id.). This plasmid, called pCS12G7, carries an active tcpAgene as shown by the following experiments:

a. When pCS12G7 is introduced into the tcpA mutant strain RT110.21, itcomplements its pilus defect and the plasmid carrying strain once againproduces the TcpA pilus.

b. DNA sequencing of the entire tcpA gene confirmed its location onpCS12G7. This sequence is presented in the FIGURE.

c. When introduced into V. cholerae strains 359-N1 or 569B-N1, pCS12G7induces a hyper-piliation phenotype in which these strains produce 2-3times more TcpA pill than usually produced in expression media. 569B-N1isan Inaba serotype strain of V. cholerae. It carries a deletion in thectxA gene but is ctxB+. The strain was constructed by transduction ofthe ctxABKm^(r) mutation of 0395-NT into a spontaneously highly motilederivative of 569B (strain 569B is usually non-motile). The transductant(strain 569B-NT) was then converted to 569B-N1 by recombination withpJM290.2 (Id.).

d. When introduced into E. coli or Salmonella typhimuruim LB5000,pCS12G7 produces a protein subunit that is identical in size andimmunological properties to the TcpA protein by Western blot analysis.

B subunit of cholera toxin

The B subunit is present in cholera toxin at a ratio of 5:1 with Asubunit.It has no known toxic activity and is useful as a component forvaccines against cholera. Strains of V. cholerae having mutations whichinactivate the A subunit are known. The level of both A and B subunitscan be markedly increased by growing V. cholerae cells in mediumsuitable for production of the TcpA pilus, as described above. By usingmutants having inactive A subunit e.g., ctxA deletion strains (Id.), itis possible to grow cells to produce large amounts of both the B subunitand TcpA pilus simultaneously.

The reason for enhanced production of B subunit and TcpA pilus appearsto be that the genes encoding these proteins are regulated by the samegene--toxR--the regulation or activity of which in turn is affected bygrowth conditions.

Furthermore, by using a tcpA-specific hybridization probe (a DNAfragment containing only nucleotides known to encode amino acids of theTcpA protein), we have shown that the tcpA gene is present in allstrains of V.cholerae isolated from patients with cholera. In contrast,several environmental strains of V. cholerae (i.e., strains isolatedfrom water sources, rather than patients), do not contain the tcpA gene.These observations support the contention that tcpA encodes thecolonization factor for all V. cholerae strains implicated in humandiarrheal disease. In addition, environmental strains of V. choleraewhich do not contain thetcpA gene are known to colonize human volunteerspoorly and induce little if any immunity in those volunteers. Levine etal., In Acute Enteric Infections in Children, Ch. 26., 1981 Elsevier.The explantation for this is that those strains cannot make the TcpApilus and therefore cannot colonize humans or induce protectiveantibodies that react with the TcpA pilus.

Cholera Vaccines

The ability to produce both TcpA pilus and large amounts of B subunitmake possible the construction of a variety of cholera vaccines.

a. Killed whole cell vaccine

For this vaccine V. cholerae cells are grown under conditions that allowhigh expression of the TcpA pilus and/or the B subunit of cholera toxin.We have established such conditions for strains 0395-NI, 569B-NI, andtheir derivatives (see Example 2 below) but have shown that theseconditions can be similarly established for theoretically any strain ofV.cholerae provided TcpA and cholera toxin B subunit production are usedto optimize growth parameters. Similarly, mutant strains of V. choleraecan be isolated that produce TcpA and B subunit under growth conditionsdifferent from those presented here (see strain 569B htx-5 in example 2below). The essential aspect of our invention is that cultivation of V.cholerae under certain growth conditions can produce cells that expressTcpA pill on their surface at high level (i.e., greater than 1% of totalcell protein) and thus form a suitable cholera vaccine. Once appropriatestrains are grown under appropriate conditions, the cells are thenkilled by standard procedures, such as by treatment with glutaraldehydeor formalin. These treatments must be such as to preserve the antigenicproperties of the TcpA pilus and B subunit.

This vaccine can be supplemented with purified B subunit produced bystandard procedure, or by growing cells under conditions which enhanceitsproduction, and then purifying the protein by standard procedure.

EXAMPLE 2

This example demonstrates the production of cholera B subunit andpillared V. cholerae cells for use in killed whole cell vaccine or forpreparation of purified TcpA pilus.

At least three different strains of V. cholerae can be used forproduction of pillared cells and the B subunit of cholera toxin. Strains0395-N1 and its hyperpiliated derivative 0395-N1 (pCS12G7) are Ogawa inserotype whilestrains 569B-N1 and its hyperpiliated, hypertoxigenicderivative 569B-N1 htx-5 are Inaba in serotype. Strain 569B-N1 htx-5 isa derivative of strain 569B-N1 carrying a htx mutation isolated bystandard methods (Mekalanos, Proc. Natl. Acad. Sci. U.S.A. 75:941). Wehave found that in addition to causing hyperproduction of cholera toxin,the htx mutation also causes hyperproduction of the TcpA pilus underboth the growth conditions outlined below as well as other growthconditions that are not usually permissive for TcpA production (e.g.,high pH's such as 7.5). A cholera killed whole cell vaccine shouldpreferably contain cells of both the Ogawa and Inaba serotypes andtherefore separate cultures of one Ogawaand one Inaba strain arenormally prepared. The hyperpiliated derivatives produce 2-3 times asmuch TcpA pilus as their parental derivatives becausethey either carrythe high copy number plasmid pCS12G7 that contains the tcpA gene ofstrain 0395 (i.e., 0395-N1 pC512G7) or they carry the htx mutation(i.e., 569B-N1 htx-5). All strains are cultured as described belowexcept that ampicillin is added to the medium to a final concentrationof 50 micrograms per ml for strain 0395-N1 (pCS12G7).

Starter cultures of the selected V. cholerae strain are prepared in testtubes containing 2 ml of LB-6.5 medium (10 g Trimtone, 5 g yeastextract, 5 g sodium chloride, pH adjusted to 6.5 before autoclaving) andincubated at 30° C. on a roller incubator at a speed of 30 RPM of 18 h.Starting cell density of the starter culture is low (about 10⁷ permlinoculated from a fresh agar plate culture) and after growth,autoagglutination of the bacterial cells is apparent as clumps ofmaterialeasily visible to the naked eye. The bacterial cells that areclumped are allowed to settle out of the upright stationary tube andthen collected from the bottom with a pipet. The clumped cells are usedto inoculate 6 liters of LB-6.5 contained an appropriately designedfermentation vessel that allows air to be pumped through the medium anddispersed as fine bubbles. The culture is incubated with moderateaeration (0.5-1 liter/min)at 25° C. for 24 h during which time theculture reaches stationary phase and becomes nearly completelyautoagglutinated. The clumped bacterial cells carrying TcpA pill ontheir surface are collected by centrifugation and resuspended in 600 mlof 0.85% sodium chloride, 10 mM sodium phosphate buffer pH 7.0.

These pillared V. cholerae cells are then used for preparation ofpurified TcpA pill or killed whole cells while the cell free culturesupernatant fluid is used to purify the B subunit of cholera toxin bystandard methods(Mekalanos et al., Infection and Immunity 16:789, 1977and 20:552, 1978).

It is anticipated that the method used to kill and preserve thesepiliated cells for use in the whole cell vaccine should not destroy theimmunogenicity of the TcpA pill. In this regard, treatment of thepiliatedcells for 24-48 h with 1% glutaraldehyde or 1% formalineffectively kills the organism while maintaining the immunogenicity ofthe TcpA pilus. The formulation of one oral or parenteral dose of thiskilled whole cell vaccine should preferably include at least 10¹⁰ killedpillared V. cholerae cells of the Ogawa serotype (0395-N1 or 0395-N1(pCS12G7)) and atleast 10¹⁰ killed pillared V. cholerae cells of theInaba serotype (569B-N1 or 569B-N1 htx-5).

The protective efficacy of such a vaccine can be further improved byincluding 200-500 micrograms of the purified B subunit of cholera toxinprepared from the same cultures used to prepare the piliated V. choleraecells. Note that all four production strains are deleted for the Asubunitof the toxin and that the above culture conditions are optimalfor expression of both the TcpA pilus and the B subunit-these are thepreferred types of production strains and conditions.

Alternatively, the piliated cells above could be sheared and the TcpApiluspurified by differential sedimentation, or other methods, and thenused either alone or combined with the purified cholera B subunit as avaccine.

b. TcpA pilus vaccine

This vaccine is composed of at least an immunogenic fragment of TcpApilus (e.g., a fragment having at least one TcpA pilus determinant andpreferably at least two or three such determinants). The pilus can beprepared either synthetically or from whole cells grown underappropriate conditions and then purified by standard procedure, or itcan be prepared by using recombinant DNA methology. For example, asynthetic polypeptide corresponding to a fragment of the TcpA pilusamino acid sequence can be prepared by standard procedures, or a nucleicacid sequence encoding such a fragment may be expressed in an expressionsystem, and the resulting polypeptide purified. Standard procedures canbe used to identify such fragments, for example, partial deletions ofthe tcpA gene can be constructed, expressed as described above, and theefficacy of this partial TcpA product studied. Those fragments that haveimmunogenicity similar to the native TcpA pilus would be useful in sucha vaccine. Similarly, polypeptides which are cross-reactive with theTcpA pilus are suitable in this invention. (By cross-reactive is meantthose polypeptideswhich are immunoprecipitable with antibodies producedto TcpA pilus.) See generally, J. Mol. Immunol. 19:1541-1549, 1982; andJ. Mol. Immunol. 21:785-793, 1984.

This vaccine may be supplemented with killed whole cells of V. cholerae,with B subunit, or may be used to supplement existing vaccines.

Synthetic peptides also offer an inexpensive means for producing a pureimmunogen for use in vaccines. The deduced amino acid sequence of theTcpApilin (derived from its DNA sequence) provides the essentialinformation needed to design a synthetic peptide that might serve as animmunogen for raising antibodies that react with the TcpA pilus andblock its function (cell binding). Such immunogenic peptides can beidentified by a systematic approach in which non-overlapping orpartially overlapping peptides are synthesized and then antibodies areraised to each. The peptides that induce antibodies which either reactwith and inhibit binding of the TcpA pili to host cells, or actuallyprotect animals from virulent V. cholerae are then identified. Thesepeptides carry the protective epitopes of the TcpA pilus and cantherefore be used as a cholera vaccine, preferably after chemicallycrosslinking them to an appropriate immunologic carrier protein. Thecarrier protein enhances the immunogenicity of the peptide by providing"T-cell help functions." Many different proteins have been used aspeptide carriers but one of the best is cholera B subunit, or the Bsubunit of the heat-labile enterotoxin of E. coli (Infection & Immunity44:268-273, 1984).

EXAMPLE 3: TcpA-RELATED CHIMERIC PROTEIN VACCINE

In the same way that a chemically crosslinked TcpA-related peptidecarrier protein conjugate can be used as an immunogen, geneticallyderived fusion proteins can also serve as immunogens in a choleravaccine. These gene fusions are made from the structural gene for theTcpA pilus or from a synthetic DNA oligonucleotide that encodes peptidesequences related to the TcpA protein, and the gene for the carrierprotein. We have made such a gene fusion between the tcpA gene and thegene for alkaline phosphatase (phoA) of E. coli and, as described above,have demonstrated the production of a fusion protein in both V. choleraeand E. coli. These fusion proteins react with antibody raised againstTcpA pill and thereforewould probably stimulate antibodies against theTcpA pilus if the fusion proteins were used as immunogens. This geneticapproach can also be utilized to make other fusion proteins betweentcpA-related DNA sequences and genes for carrier proteins like LT-Bsubunit cholera toxin B subunit, diptheria toxin, and tetanus toxin(FEBS Letters 208:194-198 (1986)).

c. Heterlogous live vaccines

Living cells of Salmonella, E. coli or vaccinia virus, which have beenmodified to be relatively non-pathogenic are transformed, or otherwisemodified by recombinant DNA methodology, to encode TcpA pilus proteinand preferably also the B subunit of cholera toxin. These cells orparticles may be used to inoculate against cholera if they can stimulateantibody production against TcpA pilus and preferably also B subunit.Only an immunologically active fragment of either protein need beencoded by theseorganisms, and this vaccine can be used in conjunctionwith the above vaccines, or with prior vaccines.

EXAMPLE 4

This example describes the construction of a live heterologous carriervaccine expressing the TcpA pilus subunit. Several organisms includingS. typhi Ty21A (J. Infect. Dis. 131:553, 1975; Inf. & Immunity46:564-569, 1984), other Salmonella species (Nature 291:238-239, 1981),E. coli-Shigella hybrids (Inf. & Immunity 46:465-469, 1984), andvaccinia virus (Nature 311:67, 1984; Proc. Natl. Acad. Sci. U.S.A.80:7155, 1983), are potential live carrier vaccines that will not onlyimmunize against homologous diseases (typhoid fever, shigellosis, smallpox, etc.) but alsoagainst other diseases carried by the live carrierstrain and expressed by genes for the appropriate protective immunogens.Both E. coli and S. typhimurium strains carrying the plasmid pCS12G7 orrelated plasmids, suchas pCS12G10, produce a protein that isimmunologically identical to the TcpA pilin. Thus, this plasmid orderivatives of this plasmid will providethe necessary geneticinformation for live carrier vaccine strains to express TcpA-relatedimmunogens.

The method for preparation of this vaccine depends on the organism usedas the live carrier vaccine strain. For bacterial carriers, plasmidsexpressing the TcpA pilin are introduced by transformation orconjugation,using standard procedure. Such plasmids can be integratedinto the chromosome of the carrier strain to provide more stable geneticinheritance of the TcpA pilin gene. For viral carriers, the tcpA genesequence is genetically engineered to express in host (animal or human)cells that are infected with the recombinant virus.

The efficacy of a live heterologous carrier vaccine expressing the TcpApilin will be improved if it also expresses the B subunit of choleratoxin. The introduction of the ctxB gene as well as the tcpA gene intothesame strain of carrier organism is therefore preferred.

Once constructed, a live heterologous vaccine strain is compounded intoa vaccine by standard procedures and administered orally or parenterallyin a dose that is large enough to allow the organisms to multiply, butnot cause overt disease in the vaccinated host. (e.g. about 10⁶ -10¹⁰cells or virus particles per dose).

Use

The use of the above vaccines includes their inoculation into humans orother animals to prevent cholera and related infections. The vaccinescan be administered using standard procedure, preferably by oral route,but also by injection. Dosages will vary from about 10⁹ -10¹⁰ livebacteria or 10¹⁰ killed bacteria, and from 10-1000 mg TcpA pilus orBsubunit per kg animal body weight.

Deposits

The following deposits were made on Apr. 29, 1987, with the AmericanType Culture Collection (ATCC), where the deposits were given thefollowing accession numbers:

    ______________________________________                                        Deposit               Accession No.                                           ______________________________________                                        V. cholerae 0395-N1 (pCS12G7)                                                                       67396                                                   V. cholerae 569B-N1 htx-5                                                                           53613                                                   S. typhimurium LB5000 (pCS12G10)                                                                    67397                                                   ______________________________________                                    

Applicants' assignee, President and fellows of Harvard College,represents that the ATCC is a depository affording permanence of thedeposit and ready accessibility thereto by the public if a patent isgranted. All restrictions on the availability to the public of thematerial so deposited will be irrevocably removed upon the granting of apatent. The material will be available during the pendency of the patentapplication to one determined by the Commissioner to be entitled theretounder 37 CFR 1.14 and 35 USC 122. The deposited material will bemaintained with all the care necessary to keep it viable anduncontaminated for a period of atleast five years after the most recentrequest for the furnishing of a sample of the deposited microorganism,and in any case, for a period of atleast thirty (30) years after thedate of deposit or for the enforceable life of the patent, whicheverperiod is longer. Applicants' assignee acknowledges its duty to replacethe deposit should the depository be unable to furnish a sample whenrequested due to the condition of the deposit.

Other embodiments are within the following claims.

We claim:
 1. A method of producing a TcpA vaccine comprisinggrowing aculture of Vibrio cholerae cells in a growth medium wherein said mediuma) contains at least one of the following amino acids: asparagine,arginine, serine, glutamic acid, glutamine, wherein said growth mediumis maintained at a temperature of 22°-30° C. with exposure to oxygen, b)has an ionic strength equivalent to at least 50 μM NaCl, c) has a pH ofabout 6.5 or less; and d) is maintained at a temperature of 22°-30° C.with exposure to oxygen; wherein said TcpA pilus is present at aconcentration representing 1% or more of total cell protein; recoveringa TcpA antigen-containing fraction from said culture; and formulating avaccine comprising a TcpA antigen obtained from said fraction and aphysiologically acceptable carrier.
 2. The method of claim 1 in whichsaid cells are mutated or otherwise engineered to produce TcpA pilus at1% or more of total cell protein.
 3. The method of claim 1 wherein saidcells have a CTXA mutation resulting in non-toxic levels of choleratoxin.
 4. The method of claim 3 comprising formulating a live whole-cellvaccine from said culture.
 5. The method of claim 1 wherein said methodcomprises producing a whole-cell vaccine by combining whole cells fromsaid fraction with said carrier.
 6. The method of claim 5 furthercomprising killing said cells and providing said killed cells in apharmaceutically acceptable vehicle.
 7. The method of claim 1 or 2wherein said cells produce B subunit of cholera toxin and said methodfurther comprises purifying said B subunit from said cells andformulating said vaccine with said B subunit.
 8. The method of claim 6wherein said method further comprises purifying said TcpA pilus fromsaid cells in said fraction and combining said TcpA pilus with saidcarrier.
 9. A whole cell vaccine made by the method of claim
 1. 10. Amethod of making a vaccine comprising providing a cell engineered toinclude at least one expressible TcpA pilus gene, culturing said cellsuch that said TcpA gene results in TcpA pilus being present at aconcentration representing 1% or more of total protein,recovering a TcpAantigen-containing fraction from said culture; and and formulating avaccine comprising a TcpA antigen obtained from said fraction and aphysiologically acceptable carrier.
 11. A bacterial cell comprising atleast 1% total protein as TcpA, said cell lacking toxic levels ofcholera toxin.
 12. The cell of claim 11 in which said cell is a Vibriocholerae.
 13. A TcpA vaccine comprising a polypeptide in apharmaceutically acceptable carrier, said polypeptide comprisingprotective immunogenic determinant of the TcpA pilus of Vibrio cholerae.14. The vaccine of claim 13 wherein said polypeptide comprises at leasteight contiguous amino acids of the amino acid sequence shown in FIG. 1.15. The vaccine of claim 13 wherein said polypeptide comprises a TcpApilus monomer of V. cholerae.
 16. The vaccine of claim 13 furthercomprising the B subunit of cholera toxin.